Anti-Drug Antibody (ADA) positive controls generation against oligonucleotide therapeutics: Anti-Oligonucleotide antibody case study

Oligonucleotide therapeutics are generally single-stranded synthetic oligonucleotides with modifications in their structure to improve stability and PK properties. These therapeutics generally target RNA transcripts and reduce or modify the corresponding protein expression. Unlike most typical biologic drug classes such as monoclonal antibodies, the oligonucleotides are relatively small in size and contain fewer potential epitopes. In addition, the oligonucleotide therapeutics are delivered to immune restricted sites to remain locally confined. 

Despite these factors, there is a chance that these therapeutics may reach the areas where they can interact with immune system and generate antibody response. As a result, the expectation from the regulatory agencies is that the immunogenicity be tested, and data provided as part of the new drug application. This requirement necessitates the generation of high-quality anti-drug antibody (ADA) positive control, which is required for developing an ADA assay with good sensitivity to detect low levels of immunogenicity.

The factors described above, often seen as an advantage in developing a therapeutic, create significant challenges when generating a positive control.  A multitude of considerations need to be factored into such a study including host species, immunization schedule, adjuvant selection, antibody screening and purification methodology. 

Our Strategy

Our approach is based on decades of experience, literature-based research and preliminary pilot studies and has allowed us to successfully generate ADA positive controls against a variety of oligonucleotide therapeutics for our sponsors. 

The unique ontogeny of rabbit B cells[1] supports using rabbits as the host-species for generating strong antibody response against poor immunogenic antigens such as oligonucleotides. 

The oligonucleotides are conjugated to two different carrier proteins and used as immunogens.  These oligonucleotide conjugates are used in a unique immunization paradigm, specifically designed to overcome the inherent challenges described above, which involves multiple immunizations.  At regular intervals the blood samples are collected and screened against biotinylated oligonucleotides in an ELISA platform.  Based on the antibody response, the immunization strategy may be adjusted with a goal of achieving the highest antibody titers. 

Once a satisfactory titer is reached, appropriate antisera from individual animals will be pooled and oligonucleotide-specific antibodies purified using an oligonucleotide coupled affinity column.

Case Study: Anti-Oligonucleotide Antibody

To highlight the success of the above strategy, we present a case study in which Labcorp generated an ADA positive control against an antisense oligonucleotide therapeutic. 

In this project, we immunized five New Zealand white rabbits and antibody titers were determined at regular intervals.  All of the animals developed strong titers and, as expected, there was significant inter-animal variability in the magnitude of the response (see Table 1). 

Table 1 – Subset of ELISA Titer

Selected bleeds from all animals were pooled for antigen-affinity based antibody purification.  The antiserum was passed over the affinity column three separate times, with elutions occurring between each pass, and the eluate was concentrated and dialyzed.    

Labcorp’s unique and specifically designed approach yielded a high titered (See Figure 1), purified antibody, despite the poor immunogenic nature of oligonucleotides.

The specificity of the purified antibody was determined by competition assays using unlabeled anti-sense oligo, scrambled oligos and anti-sense oligo analogs. 

                                        
                                     Figure 1 – Purified Anti-Oligo Antibody Titer

Successful generation of this anti-drug antibody positive control enabled Labcorp’s bioanalytical team/third party vendors to develop assays sensitive enough to detect sub-nanogram quantities of ADA responses.  This enabled the regulatory review of the anti-sense therapeutic to advance to the next phase in clinical development. 

Identifying a partner with the appropriate knowledge and experience is critical when working with difficult targets.  Labcorp’s Antibody Reagents and Vaccines division includes PhD-level scientists who evaluate every antigen, including oligonucleotides.  Notably, these subject matter experts pay particular attention to the structure of the molecule and provide suggestions on including suitable functional groups for conjugation to carrier proteins and/or for labeling for ELISA screening purposes. 

By choosing a leading provider of custom ADA development, like Labcorp Drug Development, you can rely on strong scientific expertise and extensive capabilities in custom reagent development  and related ADA services, including development of PK, TK  and potency assays. Contact us today to learn how we can partner to achieve your goals.